Spectrally Silent Transformation in the Photolysis of Octopus

Spectrally Silent Transformation in the Photolysis of Octopus Rhodopsin Nishioku et al. (2001) Brandon Drescher BIO 603 http: //mrbarlow. files. wordpress. com/2007/08/octopus. jpg http: //upload. wikimedia. org/wikipedia/commons/thumb/a/a 1/All-trans-Retinal 2. svg/439 px-All-trans-Retinal 2. svg. png

Background • Rhodopsin ≈ 70% photoreceptor membrane protein • Lot of work on octopus photoactivation mechanism and rhodopsin in late 90 s ▫ Nakagawa et al. (1997, 98, 99, 2001) Bovine Rhodopsin: http: //www. ks. uiuc. edu/Services/Class/BIOPHYS 490 M/papers/Sakmar-minireview-2002. pdf http: //www. ks. uiuc. edu/Research/rhodopsin/rh_in_membrane_bigger. gif

• Plasma membrane • Cell body • Nucleus • Synaptic region Alberts et al. 2008

Alberts et al. 2008

http: //cas. bellarmine. edu/tietjen/Laboratories/Eye 07. gif

Background – Fringe Spacing • Spacing = element(s) present • Based on diffraction pattern/interfer ence pattern http: //www. ceravolo. com/images/sphere_hindle. jpg

Circular Dichroism • Spectroscopic measurements using left-handed polarized light vs. right-handed polarized light • Absorption differences based on structural (proteins) asymmetry • Protein structure stability • TG – alternative method for short time scales

Transient Grating Method • Femptosecond pulse laser system http: //plasmonic. net/images/image. R 9. jpg

• TG method ▫ Transient grating method ▫ Spectral and kinetic-based analysis ▫ Detection of protein dynamics with no optical absorption of the chromophore ▫ Various contributions to sort out mathematically �Thermal grating, diffusion coefficients, refractive index change, density http: //www. uni-potsdam. de/u/physik/Photonik/Gitter/grating 1. png

Methods • Sucrose floatation ▫ Isolation of membranes with sucrose gradient ▫ Sucrose monolaurate (SM 1200) �Non-ionic detergent http: //molecules. gnu-darwin. org/html/00150001_00175000/174829. png

Methods • Con-A Sepharose ▫ Affinity purification ▫ Separation of glycoproteins from cell surface bioworld 2010 http: //www. bio-world. com/images/sepharose. jpg

Methods • Laser flash photolysis system • TSP-1000 (Japanese) used for transient absorbance during photochemical reactions http: //www. unisoku. com/img-Laser. Flash. Photolysis. jpg

Hara et al. (1996) • Enthalpy and volume change using the TG method ▫ ▫ Various alkanes as solvents Beam splitter → focused through lens Varying decay rates Standard thermal grating signal for fringe spacing measurement

Scheme 1 • Chemical species • Lifetimes • “X” Nishioku et al. (2001)

Kinetics…. boooo Nishioku et al. 2001

Results • Figure 1: ▫ light-induced transient absorption changes (543. 5 nm) ▫ No absorbance changes beyond 100 μs (mesorhodopsin → acid metarhodopsin)

Results • Figure 2: ▫ Kinetics of transient grating method ▫ 840 nm probe wavelength

Some Key Notes • Probe wavelength affects refractive index change ▫ Due to population grating ▫ Thermal grating + volume grating intensities less sensitive ▫ Shorter λ’s = enhanced intensities • Blue light illumination ▫ 1800 -1700 cm-1 band region ▫ Change in carboxylic group of Asp-81? ? ?

Some Key Notes • Kinetics affected by temperature ▫ Figure 4 ▫ Expansion of microenvironment • Page 2926 ▫ “structure change is not the dynamics around the chromophore”

Since 2001…. • Studies into G-protein activation + • Truncated octopus rhodopsin ▫ Forward and reverse agonists (Ashida et al. 2004) • Len-free setup (Yamaguchi et al. 2003) • TG usage ▫ Enthalpy measurements with various solutions ▫ High harmonic emissions (Mairesse et al. 2010)

Critique • Spectral and kinetic study into protein conformational change • Use of many compounds in methods ▫ Previous works • Small time frames ▫ Figure 2 • Fairly hard to understand if not proficient in physical chemistry • Required to go back decades for clear explanantion http: //www. freewebs. com/andrej_gajic/Marine%20 Biology/O%20 Dofleini. jpg

No Questions? ? Then off to Dinner!!

References • • • Alberts, B. , Johnson, A. , Lewis, J. , Raff, M. , Roberts, K. and P. Walter 2008. Molecular Biology of the Cell. Garland Science, New York 5 th ed. Pgs. 917 -919 Ashida, A. , Matsumoto, K. , Ebrey, T. G. and M. Tsuda 2004. A purified agonist-activated G-protein coupled receptor: truncated octopus acid metarhodopsin. Zoological Science 21: 245 -250 Hara, T. , Hirota, N. and M. Terazima 1996. New application of the transient grating method to a photochemical reaction: the enthalpy, reaction volume change, and partial molar volume measurements. Journal of Physical Chemistry 100: 10194 -10200 Mairesse, T. , Dudovich, N. , Zeidler, D. , Spanner, M. , Villeneuve, D. M. and P. B. Corkum 2010. Phase sensitivity of high harmonic transient grating spectroscopy. J. Phys. B: At. Mol. Opt. Physc. 43: 065401 Nishioku, Y. , Nakagawa, M. Tsuda, M. and M. Terazima 2001. A spectrally silent transformation in the photolysis of octopus rhodopsin: a protein conformational change without any accompanying change of the chromophore’s absorption. Biophysical Journal 80: 2922 -2927 Nakagawa, M. , Kikkawa, S. , Tominaga, K. , Tsugi, N. and M. Tsuda 1998. A novel photointermediate of octopus rhodopsin activates its G-protein. FEBS Letters 436: 259 -262 Ostrom, R. S. and X. Liu 2007. Detergent and detergent-free methods to define lipid rafts and caveolae. Methods in Molecular Biology 400: 459 -468 Paulsen, R. , Zinkler, D. and M. Delmelle 1983. Architecture and dynamics of microvillar photoreceptor membranes of a cephalopod. Experimental Eye Research 36: 47 -56 Penelope the chipmunk – NGM ‘yourshot’ Yamaguchi, M. , Sawada, T. , Katayama, K. and M. Fujinami 2003. Development of lens-free transient grating method and its application. Nippon Kagakkai Koen Yokoshu 83: 410